Analytic device

ABSTRACT

An analytic device comprising a device housing, a dock to receive a camera enabled mobile electronic device, such as a smartphone and other smart devices, and a processing device to communicate with the mobile electronic device and to control a condition of the assay tube, such as temperature. In another example, the analytic device comprises a device housing and a circuit board. A processing device, a heating block defining a recess to support assay tube, and a resistive heater are surface mounted to the circuit board. A light source and a fan are also provided. A dock may be provided to support a mobile electronic device. The mobile electronic device communicates with the processing device to cause the application of reaction conditions to the assay tube, to perform a PCR procedure, for example. Methods are also disclosed.

RELATED APPLICATION

The present application is a continuation of U.S. patent applicationSer. No. 14/159,844, which was filed on Jan. 21, 2014, now U.S. Pat. No.9,579,655, which claims the benefit of U.S. Provisional PatentApplication No. 61/754,472, which was filed on Jan. 18, 2013, both ofwhich are assigned to the assignee of the present invention and areincorporated by reference herein.

FIELD OF THE INVENTION

Embodiments of the invention relate to analytic devices and, moreparticularly, to an analytic device including a docking station toreceive a camera enabled, mobile electronic device, such as asmartphone.

BACKGROUND OF THE INVENTION

Assay instrumentation enables the interrogation of biological andchemical samples to identify components of the sample. The sample may beprocessed prior to performing the assay. The processed sample or assaymay be placed in an assay tube and positioned in an internal compartmentof a device for performing the assay and obtaining the results. Theassay procedure may include the application of light, heat, enzymes,etc. The instrumentation includes computing and display components. Thecomputing system controls the instrumentation and processing of gathereddata. The display provides a graphical representation of the measureddata. The cost and bulk of such instrumentation systems, such as medicaldiagnostic equipment or genetic testing equipment, commonly requiresthat biological samples be shipped to a testing facility for processingand analysis. This delays the receipt of test results, often by severaldays.

The nucleic acids DNA and RNA may be extracted from a biological samplein accordance with the Boom method or modifications thereof, forexample, as is known in the art. In accordance with the Boom method, abiological sample is lysed and/or homogenized by mixing the biologicalsample with detergent in the presence of protein degrading enzymes. Thechaotropic agents and silica or silica coated beads are mixed with thelysed biological sample. The chaotropic agents disrupt and denature thestructure of nucleic acids by interfering with the macromolecularinteractions mediated by non-covalent forces, such as hydrogen bonding,van der Waals forces, and hydrophobic interactions, for example. In thepresence of the chaotropic agents, water is removed from the phosphategroups of the nucleic acids, exposing them and allowing hydrophobicbonding to the silica, such as silica or silica coated beads. Protein,cellular debris, and other substances in the biological samples do notbond to the silica and are retained in the solution. The silica beadsare washed several times to remove non-nucleic acid materials, such asproteins, lipids, cellular constituents, including cellular molecules,and other substances found in biological samples. Silica coated magneticbeads may be used to assist in the separation of the nucleic acids boundto the silica coating from the solution, via a magnetic field or magnet.The nucleic acids are then eluted from the silica or silica coated beadsinto a buffer by decreasing the concentration of the chaotropic agents.The elution buffer may be pure water or Tris EDTA (“TE”) buffer, forexample.

Polymerase chain reaction (“PCR”) is a biochemical process used in assayprocedures to exponentially copy a target nucleic acid (DNA or RNA)sequence. The PCR process can be tailored to be highly specific andsensitive, allowing amplification of a low copy number sequence into adetectable quantity. The reaction requires a combination of a targetnucleic acid sequence, a DNA polymerase, a primer (short DNA sequencethat hybridizes to a target sequence complementary to the target DNA),deoxynucleotide triphosphates (“dNTPs”) (which are joined by thepolymerase to the copied sequences), and a buffer solution includingdivalent cations (magnesium or manganese ions). The reaction proceeds intemperature cycles including: 1) a melting/denaturing stage during whichthe reaction mixture is brought to a relatively high temperature atwhich double stranded DNA separates into single strands; and 2) a lowerannealing temperature, at which the primers attach to a complementarysequence and the polymerase join the dNPT to the 3′ end of the primer,forming a complimentary copy of the sequence. This copy can then act asa template for subsequent reaction cycles. Additional heating andcooling steps may be provided to optimize the process. The highsensitivity of PCR allows use in a diagnostic assay for detection of apathogen without culturing, as may be required in alternative assays.The high sensitivity also reduces false negatives. The high specificityof PCR reduces false positives.

Quantitative Real-Time PCR (qPCR) is the real time detection of anamplified DNA or RNA sequence. This process can use intercalating dyesthat fluoresce when exposed to an excitation wavelength after the dyebinds to double stranded DNA. Alternatively, other chemistries areavailable, such as linear probes. Probe chemistries add another layer ofspecificity because specific hybridization between the probe and atarget nucleic acid sequence is required to generate fluorescence.

One example of a linear probe is a hydrolysis probe, which are nucleicacid sequences that include a reporter dye, such as a fluorophore, onthe 5′ end, and a fluorescent quenching moiety agent on the 3′ end. Sucha probe generally relies on the 5′-3′ exonuclease activity of TaqPolymerase. The fluorescent quenching of the 5′ fluorophore requiresthat the quenching agent be in proximity of the 5′ fluorophore. Thepolymerase hydrolyzes the 5′ fluorophore during the extension phase of aPCR cycle, the fluorophore is removed from proximity to the quenchingagent, allowing fluorescence from the dye to be detected. As in otherreal-time PCR methods, the resulting fluorescence signal permitsquantitative measurements of the accumulation of the product during theexponential stages of the PCR.

Another probe chemistry that can be used are structured probes, such asmolecular beacons. Molecular Beacons consist of a hairpin loop structurethat is complementary to the target sequence and a stem complementary tothe termini. One end of the termini contains a reporter dye and theother end contains a quencher dye which are brought in close proximitywhen the probe is in the hairpin state. Upon binding to its target thehairpin is opened and the fluorophore and quencher are separated,resulting in increased fluorescence. If the target sequence does notexactly match the Molecular Beacon sequence, hybridization and thereforefluorescence will not occur because the hairpin state isthermodynamically favored over the hybridized state.

qRT-PCR (Real Time quantitative Reverse Transcription PCR) enablesreliable detection and measurement of RNA targets, such as mRNA and RNAviruses. An initial cycle of the reaction employs a reversetranscriptase to make a DNA copy from an RNA template. The copies of theDNA sequence are then amplified as with conventional PCR.

The functionality of personal electronic devices, such as smartphonesand tablets, for example, is expanding. For example, smartphones arecapable of wireless data transmission, global position tracking, imageand video capture from front and rear facing cameras, data processing,data storage (including image storage), data display, time and datetracking, and acceleration measuring, for example.

Smartphones have been used in conjunction with medical devices for datacollection and analysis. For example, AliveCor, Inc., San Francisco,Calif., provides an iPhone 4, 4S, and 5 case with a built-in heartmonitor that enables performance of an electrocardiogram (ECG). TheiPhone ECG can be used by consumers, for clinical diagnostics and inveterinary applications. iBGStar (R), available from the Sanofi—AventisGroupe, Frankfurt, Germany, provides a glucose meter for diabetics thatplugs into the bottom of an iPhone. Mobisante, Inc., Redmond, Wash., hasdeveloped a handheld, smartphone-enabled ultrasound imaging device.CellScope, Inc., San Francisco, Calif., developed a smart-phone enabledotoscope for remote diagnoses of ear conditions, such as pediatric earinfections. Tinke, available from Zensorium, Singapore, monitors pulse,respiration, and blood oxygen levels. An iPhone App also displays pulse,respiration, and blood oxygen measurements, as well as composite scorerelated to fitness and wellness of a user.

SUMMARY OF THE INVENTION

In accordance with an embodiment of the invention, an analytic deviceincludes an analytic unit and a docking station or dock to temporarilyor permanently hold a camera enabled, mobile electronic device. Thedocking station or dock is a support that may be attached to an externalportion of the analytic unit, may be a part of the housing of theanalytic unit, or may be internal to the analytic unit. The dockingstation and/or the analytic unit have a window positioned so that whenthe mobile electronic device is held in the docking station, a camera onthe camera enabled mobile electronic device, such as a back facingcamera, is aligned with the window in order to capture images within inthe interior of the analytic unit. An assay tube holding an assay to beanalyzed is supported within the analytic unit. The assay tube ispositioned within the analytic unit so that the mobile electronic devicecan capture images of the assay within assay tube when the mobileelectronic device is supported by the docking station. The assay tubemay be supported in a controllable assay chamber that allows the sampleto be subjected to reaction conditions, such as heating and cooling, forexample, prior to image capture. The application of reaction conditionsmay be performed by the analytic device under at least partial controlof the mobile electronic device. The camera enabled mobile electronicdevice may be a smartphone, tablet, or iPod®, or other such smart devicethat includes at least one camera. The reaction conditions may define aPCR procedure to identify the presence of a target nucleic acidsequence, such as a nucleic acid sequence of a target virus or bacteria,for example.

In one embodiment, a sample heater is positioned within the housing toheat the assay tube and the assay within the assay tube. The heater maybe a hot air heater including a fan to blow the hot air toward the assaytube. In another example, the heater is a heating block that is heatedby resistive heating and defines one or more recesses to support one ormore assay tubes, respectively. In another example, individual heatingblocks are provided for each assay tube. In this example, individualresistive heaters may be provided for each heating block. Individualtemperature sensors may also be provided for each heating block. A fanor blower may be provided to cool the heating block or blocks.

Also within the housing is a light source positioned to excite thecontents of the assay. Respective light sources may be provided for eachassay tube, for example. Respective light pipes may be provided toconvey the excitation light from each light source to each assay tube.The heating block or blocks described above define openings with respectto respective recesses to allow for excitation of each assay byexcitation light and the imaging of each assay by the camera of themobile electronic device. Increasing fluorescence as the assay issubjected to the reaction conditions, which may be recorded in theimages captured by the camera of the mobile electronic device, isindicative of the presence of the target nucleic acid sequence in theassay and the initial concentration or quantity of the target in thesample, for example.

A processing device, such as a microcontroller or microprocessor, isprovided in the analytic device to control the heating of the heatingblock/blocks and the state of the fan (on/off and fan speed, forexample), monitor the temperature sensors, receive information and/orinstructions from the mobile electronic device, and/or provideinformation to the mobile electronic device, for example. The heatingblocks, resistive heaters, temperature sensors, and/or light sources maybe surface mountable components that are surface mounted to a circuitboard, such as the circuit board to which the processing device ismounted.

Data exchange electronics, such as wireless communication electronicsand/or electrical contacts, may also be provided within the housing toallow commands from the mobile electronic device to be communicated tothe components within the housing.

In accordance with another embodiment of the invention, a method forconducting a sample assay is disclosed comprising placing an assay tubecontaining assay within an analytic device. A signal is provided from acamera enabled mobile electronic device to cause the device to subjectthe assay mixture container to reaction conditions, such as temperaturecycling. The mobile electronic device may be supported by a dockingstation attached to or part of the analytic device. A camera on thecamera enabled mobile electronic device, such as a rear facing camera,captures one or more images of the assay within the housing. The imagemay capture the fluorescence of target nucleic acids, which isindicative of the presence of the target nucleic acid sequence in theassay. The image may then be processed to generate a data set. The dataset may be stored in an electronic memory on the camera enabled mobileelectronic device and analyzed by the mobile electronic device orcommunicated to other devices, via a network, for example. The data setmay be wirelessly transferred to another device, for example.

The mobile electronic device may capture or generate other data, aswell, to associate with the image. For example, the camera may alsodetect whether an assay tube is properly positioned and/or filled,premature opening of the lid, and other potential problems. A time stampor other associated data may be generated from the camera enabled mobileelectronic device, such as location, acceleration of the analyticdevice, temperature, test protocol, etc. Acceleration of the analyticdevice, which may be determined by accelerometer or gyroscopic sensorsin the mobile electronic device, may be used in the quality control (QC)of the assay procedure to determine if the smart phone and/or theanalytic device were dropped, inverted, or impacted, for example, whichcould interfere with the test results due to displacement of the assayin the assay tubes. A warning or system check may be provided by themobile electronic device to the user if there is a problem. A frontfacing camera on the camera enabled electronic device may also be usedto capture an image of a user running the assay procedure, or scan asample container or a label on reagents to store assay information withthe collected data, while the mobile electronic device is in the dockingstation. The assay procedure may be PCR, for example.

In accordance with one embodiment of the invention, an analytic deviceis disclosed comprising a device housing and a dock on the devicehousing. The dock is configured to receive a camera enabled, mobileelectronic device. A controllable assay chamber is within the housing,configured to support at least one assay tube containing an assay to beanalyzed. The analytic device further comprises a processing deviceconfigured to communicate with the mobile electronic device and tocontrol at least one condition of the assay tube. At least one of thehousing and the dock define a window positioned such that, when a cameraenabled, mobile electronic device is received in the dock, a camera onthe mobile electronic device is positioned to capture images through thewindow.

In accordance with another embodiment of the invention, an analyticdevice is disclosed comprising a device housing, a circuit board withinthe housing, and a processing device surface mounted to the circuitboard. A surface mountable heating block defines a recess to receive anassay tube, and first and second openings. The heating block is surfacemounted to the circuit board. A surface mountable resistive heater issurface mounted to the circuit board in thermal contact with the heatingblock to heat the heating block, under the control of the processingdevice. A fan is positioned to blow air onto the heating block to coolthe heating block, under the control of the processing device. A lightsource is positioned to expose contents of the assay tube to excitationlight through the first opening, under the control of the processingdevice. The light source may be a surface mountable light source that isalso surface mounted to the circuit board. A dock may be connected to orpart of the housing to hold a camera enabled mobile electronic device,wherein, when the camera enabled mobile electronic device is placed inthe dock, a camera on the device is positioned to allow image capture ofat least a portion of the at least one assay tube, through the secondopening in the heating block.

The first and second embodiments may further comprise the camera enabledmobile electronic device supported by the docking station. The mobileelectronic device may be configured to control at least one reactioncondition, such as by controlling operation of the heating unit, thecooling unit, and/or the light source, by providing information and/orinstructions to the processing device of the analytic device. The mobileelectronic device may be configured to provide the information and/orinstructions by a second processing device under the control of an Appstored on the mobile electronic device. The App may be configured tocause the second processing device to provide instructions to run apolymerase chain reaction (PCR) procedure.

In accordance with another embodiment of the invention, a method forconducting an assay procedure comprises placing an assay tube containingan assay mixture within an analytic device, and docking a cameraenabled, mobile electronic device to the analytic device such that acamera on the mobile electronic device is positioned to record images ofthe assay within the assay tube. A signal is sent from the docked mobileelectronic device to initiate reaction conditions. The assay within theassay chamber is exposed to reaction conditions. At least one image ofthe assay within the assay chamber is captured by the camera enabled,mobile electronic device. The reaction conditions may includetemperature cycling, for example. Parameters for the reaction conditionsmay be provided by the mobile electronic device to the analytic device.The assay procedure may be a polymerase chain reaction (PCR) procedure,for example.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a front, perspective view of an embodiment of an analyticdevice with a smartphone docked to the device, in accordance with anembodiment of the invention;

FIG. 2 is a side view of internal components of the analytic device ofFIG. 1;

FIG. 3 is a partial cross-sectional, partial breakaway view of theanalytic device of FIG. 4;

FIG. 4 is an enlarged view of Section X of FIG. 3;

FIG. 5 is a front perspective view of another example of heating blockfor use with embodiments of the invention;

FIG. 6 is a perspective view of an example a compact PCR analytic devicein accordance with another embodiment of the invention, without a cameraenabled mobile electronic device in the docking station;

FIG. 7a is a perspective view of the compact PCR analytic device of FIG.8, with a smartphone in the docking station;

FIG. 7b is a rear view of the camera enabled mobile electronic deviceused in embodiments of the invention;

FIG. 8 is a front view of the PCR analytic device of FIG. 7 a;

FIG. 9 is a perspective, partial cross-sectional, partial breakaway viewof the PCR analytic device of FIG. 7 a;

FIG. 10 is an enlarged perspective view of the upper portion of theanalytic unit of FIG. 7 a;

FIG. 11 is an enlarged perspective, partial breakaway, partialcross-sectional view of the upper portion of the analytic device of FIG.9, with the lid closed;

FIG. 12 is an upper perspective view of FIG. 11, with the lid removed;

FIG. 13 is a rear perspective, partial breakaway, partialcross-sectional view of the upper portion of the analytic unit of FIG.12, with the lid removed;

FIG. 14 is a perspective view of a core portion of the analytic unit;

FIG. 15 is a perspective view of the upper portion of the core, with aplenum wall removed;

FIG. 16 is a front view of FIG. 15;

FIG. 17 is a similar view as the FIG. 15, with a heating block separatedfrom the circuit board;

FIG. 18 is a rear perspective view of the core of FIG. 17;

FIG. 19 a rear perspective view of the upper portion of the core of FIG.15; and

FIGS. 20-26 are a flowchart of an example of the operation of the PCRanalytic device of FIGS. 6-19.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In accordance with an embodiment of the invention, a camera enabledmobile electronic device, such as a smartphone, tablet, or iPod®, forexample, is used as both a controller for an analytic system and as animaging component of the analytic system. Since many users will alreadyown a smartphone, tablet, or iPod® that is able to act as the cameraenabled mobile electronic device, the cost of the system is greatlyreduced. In addition, the mobile electronic device allows communicationof collected data for remote processing. As used herein, the term“camera enabled mobile electronic device” or “mobile electronic device”is a consumer electronic device including a camera, a display screen, aprocessing device, a wireless communication component, and an inputcomponent, such as a keyboard and/or a touch screen of the display. Thecamera may be on an opposite face of the mobile electronic device thanthe input component. Such a camera is referred to as a “rear facingcamera.” The mobile electronic device may also include a front facingcamera (on the same face as the input component), global positionsensors, acceleration sensors and/or tilt (gyroscopic) sensors, amicrophone, and/or fingerprint recognition, for example. In anotherembodiment of the invention, a camera enabled processing device isintegral with the analytic unit. The analytic unit may be configured toperform PCR, for example, to identify the presence of a target nucleicacid sequence in an assay. The target nucleic acid sequence may be froma virus or bacteria, for example. Additional applications are discussedbelow.

FIG. 1 shows an example of a desktop PCR analytical device 10 comprisingan analytic unit 12 and a docking station or dock 14, in accordance withan embodiment of the invention. In FIG. 1, a camera enabled mobileelectronic device 16 is supported in the dock 14. The camera enabledmobile electronic device 16 includes a display screen 18, which may be atouch screen, for the input of instructions. The input of instructionsmay include the selection of pre-determined assay routines, such as oneor more PCR routines, stored on the mobile electronic device. In thisexample, the mobile electronic device 16 also includes a front facingcamera 20 and a speaker 21. A speaker is provided at the bottom of themobile electronic device 16, as well. The front facing camera 20 may beused to capture an image of a code on an assay tube or an assay kit, forexample, a code on a reagent container, an image of the user, etc.,while the mobile electronic device 16 is supported in the dockingstation. A rear facing camera 52 shown in FIG. 2, could be used tocapture such images prior to docking in the docking station 14. Thisinformation may be attached to either a data set or an image from anassay, as discussed below. The speaker 21 can be used to alert a user toan error condition and provide voice instructions for performing theassay, for example.

The analytic unit 12 comprises a housing 22 defined by multiplerectangular walls. The top wall 24 of the housing 22 may comprise ahinged lid that rotates about a hinge (not shown) to open and enableaccess to the interior of the analytic device 12. A more detailedexample of a hinged lid is discussed below with respect to the secondembodiment of the present invention. The housing 22 may comprise heatresistant plastic, for example.

FIG. 2 is a view of the PCR analytic device 10 of FIG. 1, with thehousing 22 and the docking station 14 removed. The mobile electronicdevice 16 is shown in proper relation to the internal components, as ifthe dock 14 were present. An internal frame 30 directly or indirectlysupports the internal components of the analytic unit 12 and the housing22.

An assay tube 46 containing an assay 48 is shown above an assay tubeholder 50. An assay tube 46 is shown in position in the assay tubeholder 50, as well. The assay 48 may comprise a mixture of isolatednucleic acid and reagents. Any standard, off the shelf PCR tubes 46 maybe used. For example, the assay tube 46 may be a 0.1 ml or 0.2 ml PCRtube, or other thin-walled commercially available PCR tubes. SuitablePCR tubes may be obtained from Phenix Research Products, Candler, N.C.,for example. The assay tube 46 may be positioned by opening the hingedlid 24, allowing insertion of the assay tube 46 into the assay holder50. Insertion of the assay tube 46 into the assay holder 50 is indicatedschematically by the arrow A.

The assay tube holder 50 in this example is bolted to the frame 30 bybrackets 53, two of which are shown in FIG. 2. The assay tube holder 50and the frame 30 may also be molded plastic and the two components couldbe molded in one piece.

The mobile electronic device 16 is positioned by the dock 13 so that therear facing camera 52 (shown in FIG. 3) of the mobile electronic device16 is positioned to capture images through a transparent heat shield 54.The assay holder 50 defines an open region that allows both heated airand cooling air to circulate about the assay tube 46, and allows lightto both excite the assay in the container and be emitted from the assayfor detection by the rear facing camera 52.

A blower 60 comprising a fan blows air through a first open end 62 a ofa compartment 62, allowing controlled heating and cooling of the assay48 in the assay tube 46, through the second open end 62 b of thecompartment. The compartment 62 includes a heating coil 64, shown inFIG. 3, which heats air blown through the compartment. The heating coil64 is activated by applying a voltage to the coil to cause current flow,for example. The compartment 62 is attached to the frame 50 by a bracket66, two of which are shown in FIG. 2.

FIG. 3 is a side, partial cross-sectional view of FIG. 2. The rearfacing camera 52, is positioned in the dock 14 it faces the transparentheat shield 40 and is able to the image the assay tube 46 held by theassay holder 42. The heating coil 64 in the compartment 62 is shown. Theblower 60 has an intake 68 through which air is drawn into thecompartment 62 when the blower is operating.

Also shown in FIG. 3 is a circuit board 70 and a power supply 82 mountedto the circuit board. The power supply 82 may include battery and/or avoltage converter if the analytic device 10 is to be plugged in to linevoltage through a socket in a wall, for example. A processing device 84,such as a microcontroller or microprocessor, for example, showndistanced from the circuit board 70 in FIG. 3, is also mounted to thecircuit board. A light source 86, such as light emitting diode (“LED”),is also shown mounted to the circuit board 70, below the assay holder50. The LED acts as an excitation source to excite fluorophores andother markers or dyes in the assay. Memory and other electronic devices(not shown) may also be mounted to the circuit board 70. The circuitboard 70 may be connected to the assay holder 50 and to the internalframe 30. Examples of processing devices that may be used are discussedbelow.

FIG. 4 is an enlarged view of Section X in FIG. 3. The LED 86 directsexcitation light though a light pipe 88. The light pipe 88 allowsinternal reflection of the light from the LED 86 to convey the light tothe assay tube 46 and assay 48. The light pipe 92 may be an opticalfiber, for example. The heating coil 64 contained within the compartment60 heats air that is blown by the fan 70 through the heating coil 64.The heated air is blown past the assay tube 46, heating the tube andassay 48. When the heating coil is not activated, the air blown by theblower 60 through the compartment 62 cools the assay tube 46. Atemperature probe 90 detects the temperature of the air stream flowingthrough the compartment 60.

An excitation filter 92 may be placed between the LED 86 and the lightpipe 88 to remove light emitted by the LED with a wavelength thatoverlaps with the wavelength of the assay dye emission. The excitationfilter may also be provided between the light pipe 88 and the assay tube46. In either case, by filtering the light from the LED 86 by the filter92, the light detected by the camera 52 of the mobile electronic device16 in the emission band of the dye will be from the dye, not be from theLED 86. For example, if the LED is providing light in a blue wavelengthband and the emission dye emits light in a green wavelength band, thefilter will remove green light from the light provided by the LED. Anemission filter (not shown) may also be provided between the assay tube48 and the camera to remove light emitted by the LED with a wavelengththat overlaps with the wavelength of the light provided by the LED. Inthis example, it would filter blue light. In this way, the detectedlight will not be from the LED. An example of an excitation filter andan emission filter are described with respect to the second embodiment.The filters used for a particular assay may depend on the emission dyeand LED used.

The temperature probe 90 may also be mounted to a circuit board 70. Theprocessing device 84 controls operation of the heating coil 64, theblower 60, and the LED 86, and receives signals from the temperatureprobe 90. In particular, the heating coil 64 and the blower 60 arecontrolled by the processing device 84 to heat and cool the assay tube46 and the assay 48 to desired temperatures and to maintain thetemperatures within desired ranges for desired time intervals duringrespective assay procedures. The LED 86 is turned on at appropriatetimes by the processing device 84 to excite the fluorophores and/orother reactants in the assay 48, while the camera 52 images the assay.

Instead of the heating coil 64, a heating block 150 may be used, asshown in the perspective view of FIG. 5. In this example, the heatingblock 150 also functions as the sample holder. The heating block 150 isa solid block of metal or metal alloy, such as aluminum or an aluminumalloy, for example, which is mounted to the frame 30. The heating block150 may comprise aluminum alloy 6061, for example.

Five recesses (shown in phantom) 152 are defined in the heating block150. Five assay tubes 46 are shown in the five respective recesses 152.More or fewer recesses may be provided. Tube covers 46 a closing eachassay tube 46 are shown above the recesses 152. A resistive heatingelement 154 extends through a length of the block 150, allowingcontrolled heating of the assay tubes 46 and the assays 48, under thecontrol of the processing device 82. Fins 156 are provided to improveheat dissipation. A fan or blower (not shown), such as the blower 60,may be positioned to face the fins 156 for faster cooling of the fins156 and the heater block 150, decreasing temperature transition times.The compartment 62 shown in FIGS. 2-4 is not needed when the heatingblock 150 is used. The use of the heating block 150 provides a morecompact footprint than use of the extended compartment 62 and enablesheating a number of assay tubes 46 at the same time.

The heating block 150 also defines one or a plurality of openings 158 infront of the recesses 152 to allow for imaging of the assays 48 in theassay tubes 46 by the camera 52. Openings are also provided in thebottom of the heating block 150 (not shown) to allow excitation light toilluminate the assays 46. The heating block 150 may be used in the firstembodiment describe above, or in the second embodiment described below.

Assay Example

In the following example, the PCR analytic device 10 in accordance withthe first embodiment or the second embodiment discussed below, is usedin an assay procedure to observe or identify one or more nucleic acidsequences in an assay. It would be apparent to one of ordinary sill inthe art that aspects of this example are also applicable to the secondembodiment, described below. The assay may be derived from a human orother animal sample, or a plant sample, for example. The assay may alsobe derived from an environmental source, such as a water supply, orsoil, for example. In one example, the source may be human blood orother tissue sample, for example. Nucleic acid sequences of viruses,bacteria, fungi, protozoa, or invertebrate parasites may be identifiedin a sample.

The PCR analytic device 10 may also be used in determining humanidentity, in paternity testing, forensics, defense and homeland securityto detect bio-weapons, anti-counterfeiting, plant breeding, foodtesting, genetically modified organism (GMO) testing, and veterinarytesting, for example, as well as in research and education. Examples ofparticular applications include testing for disease vector organisms,such as a mosquito carrying West Nile Virus; a patient sample in aremote location, such as a test for viral disease; or a livestock borneillness, such as blue tongue in cattle.

Examples of assays that may be performed by the analytic unit 12include, real-time PCR, immuno PCR, DNA melting curve analysis, and DNAmicroarrays, for example.

Step One: Scan Kit

In one example, the user places a mobile electronic device 16 in thedocking station 14, selects a sample prep kit, and scans a barcode onthe kit with the front facing camera 20 of the mobile electronic device16. A sample prep kit may contain salts, buffers, divalent cations,nucleotides, polymerase enzymes, and linear probes including reporterdyes for identifying one or more target nucleic acid sequences, forexample, as is known in the art and discussed above. The reagents may belyophilized. The one or more target nucleic acid sequences may beindicative of the presence of one or more viruses, bacteria, or othersources of nucleic acid, as discussed above, for example. Sample prepkits for particular nucleic acid sequences are commercially available.

As noted above, if the mobile electronic device 16 has not yet beenplaced in the dock 14, the rear facing camera 52 may be used. Asmartphone 16 will be referred to in this example. Operations of thesmartphone 16 are controlled by a processing device, such as amicroprocessor, for example, under the control of software, such as aPCR App, for example. PCR Apps may be provided by or downloaded fromBiomeme, Inc., Philadelphia, Pa. The App may cause the processing deviceof the smartphone 16 to automatically create a new sample profile,generate a unique ID to link the assay with a patient profile, and linkthe assay with subsequently generated sample, for example. The sampleprep kit identification could also be downloaded to a centralized serverthat could, for instance, track the use of assay kits, inform a userabout the number of kits remaining, and/or automatically order new kitsif needed.

In addition, the touchscreen on the display screen 18 of the smartphone16 allows input of patient data, such as age, sex, presence of fever,and other symptoms, for example. Some data may be auto-populated by thesmartphone 16, such as time and date, GPS coordinates, and climate data,for example, under the control of the PCR App.

Step 2: Sample Preparation

A user obtains a test sample, such as a blood or urines sample, in aconventional manner. Nucleic acids may be isolated from the patientsample by any method known in the art, such as the Boom method,discussed above. Pre-isolation processing may be required for certainsamples, as is known in the art.

A solution containing isolated nucleic acid sample is introduced into anassay tube 46 containing the reagents in the PCR kit, in lyophilizedform. A lid of the assay tube is closed and the tube mixed. Thelyophilized reagents in the assay tube 46 dissolve in the solution.

Step 4: Thermal Cycling and Image Capture

In one example, the user places the assay tube 46 containing the assay48 into the assay holder 42 in the device 12. The smartphone 16 dockedin the docking station 14 communicates with the processing device 84 ofthe PCR device 10, via the PCR App. The PCR App may also cause a Startbutton to be displayed on the display screen 18, which may be pressed tostart the assay procedure. The assay is heated and cooled at differenttemperatures for predetermined periods of time defined by the PCRprotocol being run by the PCR App. At specific times during the assayprocedure, the assay is excited by excitation light and images arecaptured through the viewing window by the back facing camera 52 of thesmartphone 16. Increasing fluorescence is indicative of amplification ofthe target nucleic acid sequence. If the particular target is notpresent, fluorescence will not increase.

The images may be compared to other captured images or processed by theprocessing device of the smartphone 16 to extract luminosity data thatcan be used to determine if the assay shows increased fluorescence, forexample, via the PCR App. The data and/or image may then be stored andthe image deleted, if desired, to preserve system memory. The images maybe displayed on the display screen 18. Alternatively, the processedluminosity values may be displayed on the screen 18, or simply a finalresult (target present or not present, for example), may be displayed.An example of a thermal cycling/image capture procedure is described inmore detail, below.

The App on the smartphone 16 may be configured to cause the processingdevice of the smartphone 16 to encrypt the assay results, store them onthe smartphone 16, and/or upload them to a database, such as a clouddatabase, if/when the smartphone 16 is within a cellular network, Wifizone, or other wireless protocol. If the network or Wifi is notavailable, results may be saved on the smartphone 16 until cellular orWifi access is available. Uploading to a database preserves memory onthe smartphone 16 and/or the analytic device 10. The results may also beautomatically sent to the contact information for the patient and/orcaretaker, if desired or previously selected.

The patient may also receive or be provided with an option to receiveeducational information by their own smartphone or by email about theblood borne pathogen they may have been diagnosed with, to educatethemselves and learn how to mitigate the severity of the infection, aswell as symptoms that might indicate they need to go back and receivefollow up care. The patient may be free to access the results via theirown smartphone or via a website, along with any doctor or clinicianlinked with the patient who will be able to advise on further treatment.

Step 5: Disposal

At this point the user is free to dispose of the assay tube 46 and moveonto the next test, to start the process again.

In another application, the PCR analytic device 10 may be used toidentify DNA tags, which are self-contained authentication labelsincluding a mix of oligonucleotides that provide a unique signal whenappropriately interrogated. In the case of anti-counterfeiting, the PCRanalytic device 10 acts as a DNA reader, which interrogates a DNA tag todetermine its signal and make the association with a unique or knownclass of signals. DNA tags of a unique sequence can be placed into inks,paints and pharmaceuticals, for example, to create a unique ID fortracking and verifying throughout the supply chain, as is known in theart. One way to read the unique DNA tag is with and primerscomplementary to the unique DNA tag sequence.

The DNA tag sample collection may comprise dissolving the ink or paintto release the DNA and then capturing that DNA via nucleic acidextraction technologies. Once the pure DNA is isolated it may be placedin an assay tube 46 and a PCR thermal cycling reaction may be run by theanalytic device 10, as described above.

In accordance with a second embodiment of the invention, a more compactPCR analytic docking station 200 than the embodiment of FIG. 1 isdescribed with respect to FIGS. 6-19. It will be apparent to one ofordinary skill in the art that aspects of the second embodiment areapplicable to the first embodiment and aspects of the first embodimentare applicable to the second embodiment.

FIG. 6 is a perspective view of an example the compact PCR analyticdevice 200 in accordance with the second embodiment. The device 200comprises an analytic unit 202 and an external docking station 204 for acamera enabled mobile electronic device 206 (not shown in FIG. 6), suchas a smartphone or tablet, for example.

FIG. 7a is a perspective view of the compact PCR analytic device 200 inaccordance with the embodiment of FIG. 6, with a smartphone 206 in thedocking station 204. The smartphone 206 includes the display 18, thefront facing camera 20, the speaker 22, and the rear facing camera 52discussed above with respect to the mobile electronic device 16 used inthe first embodiment. FIG. 7b is a front view of the rear face of thesmartphone 206, showing the rear facing camera 52 and a light source 53,such as a flash. The rear facing camera 52, as well as the front facingcamera 20, are typically CMOS sensors.

In one example, the analytic unit 202 may have dimensions of about 13.5cm×4.1 cm×5.8 cm. The analytic device 200 in this example has a weightof about 15.8 ounces with the smartphone 206 in the docking station 204and about 11.5 ounces without the smartphone in the docking station 204.The PCR analytic device 200 may be handheld and/or may sit on a desk,for example. The size of the analytic unit 202 may vary. A handheld unitin accordance with embodiments of the invention weighs less than twopounds.

Returning to FIG. 6, the docking station 204 comprises a flat supportingplate 208 and a rim 210 extending from an edge 212 of the flat plate.The rim 210 may be configured to receive the smartphone 206 in a snapfitor a pressfit, for example. In this example, the smartphone 206 isretained in a snap-fit by flexible sections 210 a-210 d of the rim 210that snap over the smartphone 206 when it is inserted into the dockingstation 204. An extended section 211 defined by slots 233 through therim 210 extends from an edge of the rim, slightly over the smartphone206, as shown in FIGS. 6-8 and 10, for example. The rim 210 comprisesrim sections 210 a-210 d that extend partially around the edge 212 ofthe flat plate 208, to allow access to control buttons and ports on theedge of the smartphone 206, as shown in FIG. 8.

In a pressfit, the docking station 204 is configured so that thedimensions of the station, as defined by the location of the rim 210, isabout the same as that of the smartphone 206 so that the rim bearsagainst the sides of the smartphone when the smartphone is in thedocking station. The docking station 204 may have other configurations,as well. For example, the docking station 204 may have a front face andthe mobile electronic device 206 may be inserted into the dockingstation through a slot at the front or side of the station, for example.The front face of such a docking station 204 may be the front wall ofthe analytic unit 202 and the docking station can be interior to theanalytic unit, for example.

FIG. 8 is a front view of the docking station 204. In this example, twoholes 214, 216 are provided through the flat plate 208. One hole 214 ispositioned to be aligned with the rear facing camera 52 of thesmartphone 206. The second hole 216, which is optional, is aligned withthe light source 53 to enable illumination by the flash of thesmartphone 206, for calibration and for setting the ISO, the shutterspeed, the white balance, and sensitivity settings of the camera 52. Thesecond hole 216 is optional. The bottom of the docking station 204 mayinclude extensions 218 to tilt the analytic device 200 when the devicerests on a flat surface, such as a table or desk, for example, tofacilitate use of the touchscreen on the smartphone 206.

An optical filter 215 may be provided in the first opening 214 (or acorresponding opening in the wall of the analytic unit 202) to limitpassage of light below a predetermined wavelength, as discussed furtherbelow. The optical filter 215 acts as an emission filter that removeslight emitted by the LED. For example, if the LED emits blue light, thefilter removes blue light. In this way, the detected light will be thelight emitted by the emission dye, not the light emitted by the LED.Examples of filters 15 that may be used include Wratten No. 15 gelfilter that blocks wavelengths of 510 nm and below, or a Wratten No. 16gel filter, that blocks wavelengths of 520 nm and below, (not shown)from The Eastman Kodak Co., Rochester, N.Y.

A connector 213 may be provided in the docking station 204 for thesmartphone 206 to plug into, to provide direct electrical connectionbetween the smartphone 206 and the analytic unit 202. A power supplyport of an iPhone® may be connected to the connector 213 via a USB orother serial hardware connector, such as Apple® Lighting® connector, forexample. The mobile electronic device may also communicate with theanalytic device wirelessly, such as via Bluetooth wireless technology,for example.

Returning to FIGS. 6 and 7, the analytic unit 202 comprises front andrear rectangular walls 220, 222, a first and second sidewalls 224, 226,and top and bottom, rectangular walls 228, 230, respectively. The topwall 228 is a hinged lid connected to the upper edge of the rear wall222, enabling access to the upper portion of the interior of theanalytic unit, (as shown in FIG. 9, for example). The first and secondrectangular walls 224, 226 include semi-oval or semi-circulardepressions 242 beneath edges of the lid 228, to facilitate opening ofthe lid by hand. Other shaped depressions may be provided. The walls ofthe analytic device 202 and the docking station 204 may be formed by aplastic or metal by any appropriate technique, such as bythree-dimensional printing, forging, metal injection, welding, and/orcasting with electrical discharge machining, for example.

The docking station 204 may be a separate component connected to thefront rectangular wall 220 or the flat plate 208 may be formed integralwith the front wall 220. When a separate component, the docking station204 may be removably connected to the front wall 220 by a user by asliding and/or snap mechanism, for example. This enables the user toconnect a docking station 204 configured for different mobile electronicdevices having different sizes.

FIG. 9 is a perspective, partial cross-sectional, partial breakaway viewof the PCR analytic device 200 of FIG. 7, with the smartphone 206 in thedocking station 204, the first side wall 224 of the analytic device 202removed, and the lid 228 in an open position. In an upper portion of theanalytic device 202, a chamber 230 is provided between the front wall220 and a plenum wall 233. In FIG. 9, a rear assay tube 234 c is shownand a middle assay tube 234 b is shown in cross-section. A front assaytube 234 a is shown in FIG. 10 and other Figures. A blower or fan 236 ispositioned below the assay tubes 232 a, b, c. A battery 238 ispositioned below the chamber 230. A battery charging circuit 239 isbelow the fan 236. The assay tubes 234 c, 234 b are in plenum behind theplenum wall 233, as discussed below.

FIG. 10 is an enlarged perspective view of the upper portion of theanalytic unit 202 with the hinged lid 228 open, showing the tops of thethree assay tubes 234 a, 234 b, 234 c. More or fewer assay tubes 234 maybe provided. The lid 228 in this example is connected to the rearrectangular wall 222 by a hinge 240. A magnet 244 is provided in arecess in the lid 228 and a magnet 246 (shown in FIG. 9) is provided ina recess in a ledge 248 to maintain the lid in a closed position. Otherrecesses in the lid 228 are provided to decrease the amount of materialin the lid, decreasing the cost and weight of the analytic unit 200.

The interior surface of the lid 228 includes three protrusions 252 a,252 b, 252 c for bearing against the tops 254 a, 254 b, 254 c of thethree assay tube 234 a, 234 b, 234 c, respectively. In FIG. 11, whichshows the lid 228 in a closed position, the protrusion 252 b bearsagainst the top 254 b of the assay tube 234 b. In this example, theprotrusions protrude from a common base 260. FIG. 13, which is a partialbreakaway, partial cross-sectional view through the analytic unit 200with the lid almost completely closed, shows the middle protrusion 252 bin contact with the middle assay tube 234 b. The middle protrusion 252 band the middle assay tube 234 are shown cross-section. The assay tubesin this example have a conical shape, and sit in respective conicalrecesses in respective heating blocks, as discussed further below.

The protrusion 252 b in this example comprises the common base 260,which has a rectangular cross section, and the protruding section 252 b,which has a semi-circular cross-section. The other protrusions 252 a,252 c similarly protrude respective from the base 260. The base 260 isreceived within a rectangular recessed section in the lid. 262. Theprotrusions 252 a, 252 b, 252 c provide a spring force against the assaytubes 234 a, 234 b, 234 c, respectively, to improve their thermalcontact with the surfaces of the recesses in the heating blocks 282. Aspring force of from about 0.5 to about 4 pounds per square inch may beapplied, for example. The protrusions 252 a, b, c may comprise siliconerubber or other resilient elastomeric material, for example. Otherconfigurations for providing a spring-like, bearing force against theassay tubes 234 a, b, c may also be used such as coil or lead springs.

The tops 254 a, 254 b, 254 c of the assay tubes 234 a, 234 b, 234 c maybe hinged caps that rotate about a hinge on the containers duringopening and closing of the containers. The hinged caps in this examplehave edges with protrusions for engagement by a user's finger, tofacilitate opening and closing of the lid 228. As noted above, anystandard, off the shelf PCR tubes 234 a, b, c may be used. For example,the assay tubes 234 a, b, c may be 0.1 ml or 0.2 ml PCR tubes, or otherthin-walled commercially available PCR tubes, for example. Suitable PCRtubes may be obtained from Phenix Research Products, Candler, N.C., forexample.

FIGS. 10 and 11 show air exit vents 262 through the rear wall 222,behind the assay tubes 234 a, b, c. The vents 262 in this example aredefined by parallel walls 264 protruding from the rear wall 222 into theinterior of the device 202, between the vents 262, as best shown in FIG.10. Air inlet vents 263 are also provided in the side walls 224, 226 andbottom wall 230 to air to be drawn into the analytic device 202 by thefan 236 for cooling.

FIG. 13 is a rear perspective, partial breakaway, partialcross-sectional view of the upper portion of the analytic unit 200, withthe lid 228 removed. The chamber 230, shown in FIG. 11, is furtherdefined by the second side wall 226 of the device, the first side wall224 of the device (not shown in this view), and a bottom plate 272. Theopening 216 and a portion of the opening 214 through the front plate 208of the docking station, which also penetrate through the front wall 220of the analytic unit 202, are shown. The plenum wall 233 defines threeopenings 274, as shown in FIG. 14. The rear opening 274 c and a part ofthe middle opening 274 b are shown in FIG. 12. The front opening 274 ais not shown in this view. Each opening 274 a, 274 b, and 274 b ispositioned in front of an assay tube 234 a, 234 b, 234 c, respectively,as shown in FIG. 14. In the view of FIG. 12, the openings 274 c, 274 bare shown in front of the assay tubes 234 c, 234 b. Inclined walls 278,279 facilitate placement of the assay tubes 234 a, b, c.

The assay tube 234 b is received within a recess 280 b defined in aheating block 282 b. The chamber 230, which is one example is about 1.25inches from the front wall 220 to the back wall 270, enables the fieldof view of the camera of the mobile electronic device 206 to encompassall of the assay tubes 234 a, 234 b, 234 c. Below the heating block 282is a light pipe 284. The light pipe 284 has an input surface 286adjacent a light emitting diode (“LED”) 288. An output side of the lightpipe 284 is adjacent to an opening 289 defined in the heating block 282,below the recess 280. The heating block 282, the light pipe 284, and theLED 288 are mounted to a printed circuit board 300 as discussed furtherbelow.

FIG. 14 is a perspective view of a core portion 271 of the analyticdevice 202 and the fan 236 shown in FIG. 9, for example. The core has arear wall that in this example is a printed circuit board 300. Theplenum chamber wall 233 of the chamber 232 is part of core 271, in thisexample. As mentioned above, the assay tubes 234 a, 234 b, 234 c, andthe assay tube 234 a, are supported between the printed circuit board300 and the plenum wall 233. Openings 274 a, 274 b, 274 c through theplate 233, which are shown in part in FIGS. 11 and 12, for example, arealso shown.

The fan 236 in this example is also mounted to the printed circuit board300. The fan 236 comprises a front wall 302 defining an intake 304. Fanblades 306 blow air upward in FIG. 14, through an open top of the fan,to a plenum chamber behind the intermediate wall 233, where the assaytubes 234 a, b, c to cool the heating block 282 and other heating blocksdescribed below. The fan 236 may be powered by a brushless DC motor, forexample. The core 271 may be fastened to internal walls of the housingor supported by plastic bosses (not shown) in the walls, for example.

FIG. 15 is a perspective view of the upper portion of the core 271, withthe plenum wall 233 removed. Three heating blocks 282 a, 282 b, 282 c,each defining a recess 280 a, 280 b, 280 c to receive a respective assaytube 234 a, 234 b, 234 c (not shown in this view). Each heating block282 a, 282 b, 282 c also defines an opening 310 a, 310 b, 310 cextending through each heating block to the respective recesses 280 a,280 b, 280 c. The openings 310 a, 310 b, 310 c are aligned with theopenings 274 a, 274 b, 274 c, respectively, in the plenum wall 233 ofFIG. 14, for example. The chamber 230, shown in FIG. 9, for example, hasa sufficient length for the viewing angle of the rear facing camera 52to encompass the three openings 274 a, b, c, in order to image the threeassay tubes 234 a, b, c, through the openings 310 a, b, c.

Three light pipes 284 a, 284 b, 284 c are shown below each heating block282 a, 282 b, 282 c, respectively, as discussed above with respect toFIG. 15, for example. Three LEDs 288 a, 288 b, 288 c are providedadjacent to input sides of the light pipes 284 a, 284 b, 284 c, as wasalso discussed with respect to FIG. 15. The light pipes 284 a, 284 b,284 c guide the light received from the respective LEDs 288 a, 288 b,288 c by the input side of the light guide, to the output side of therespective light guide. In this example, the light provided by each LED288 a, 288 b, 288 c is reflected 90°. As shown in FIG. 13, for example,each heating block 282 a, b, c also defines an opening 289 through thebottom of each heating tube to the bottom of the recess 280 a, b, c forpassage of the excitation light from each light pipe 282 a, b, c to eachassay tube 234 a, b, c.

According to their specifications, LEDs 288 a, b, c in one example emitblue light with a brightness of 550 mcd, a power of 130 mW, and adominant wavelength of 470 nm, for example. The LEDs 288 a, b, c may beobtained from Optic Technology, Inc., Pittsford, N.Y., for example. Thelight pipes 284 a, 284 b, 284 c may comprise acrylic and may be formedby laser cutting, for example. One or more additional LEDs (not shown)may be provided adjacent each LED 288 a, b, c to emit light at anotherwavelength, in order to excite different reporter dyes and test foradditional types of nucleic acids. The assays may be excited at eachwavelength in an alternating sequence or simultaneously.

An optical filter 312 is shown between the input sides of the LEDs 288a, 288 b, 288 c and the light pipes 284 a, 284 b, 284 c. The opticalfilter 312 is an excitation filter configured to remove light emitted bythe LEDs 288 a, b, c having a wavelength that overlaps with thewavelength of assay dye emission. In this way, the light detected by thecamera of the smartphone camera will not be from the LEDS. For example,the dye may be excited by blue light and emit green light. The opticalfilter 312 is therefore configured to filter green light in the lightprovided by the LEDs 288 a, b, c. The optical filter 312 may be aRoscolux #385 Royal Blue lighting filter, available from RoscoLaboratories, Inc., Stanford, Conn., for example, which issued tocomprise a co-extruded polycarbonate film having a thickness of 0.003inches (76.2 microns). The filter 312 may be provided between the lightpipes 284 a, 284 b, 284 c instead. If additional LEDs are provided toprovide excitation light at additional wavelengths, as discussed above,the optical filter 312 may comprise additional respective sections toappropriately filter the excitation light of the additional LEDs. Thesmartphone 206 may provide separate images resulting from excitation ateach wavelength, or provide the captured data in a single images, forexample.

A slot 314 through the printed circuit board 300 is also shown in FIG.14 FIG. 15, which is a front view of FIG. 14, shows additional slots 314adjacent to and between each heating block 282 a, 282 b, 282 c. Theslots 314 insulate the heating blocks 282 a, b, c from each other,decreasing heat transfer from one block to an adjacent block, throughthe printed circuit board 300.

FIG. 17 is similar to FIG. 15, except that the heating block 282 a isshown separated from the printed circuit board 300, to show a resistiveheater 320 mounted to the board. Respective resistive heaters andthermistors are mounted to the board 300 for the heating blocks 282 band 282 c, as well. Also shown is a thermistor 322, which is alsomounted to the board 300. The assay tubes 234 a, b, c, are shown abovethe respective recesses 280 a, b, c.

The resistive heater 320 may be a high chip SC3 Series power resisteravailable from TT Electronics PLC, Surrey, England, for example. The SC3series high chip power resistors are said to have a power dissipation at70° C. of three watts, a resistance range of 1RO to 10K, and an ambienttemperature range of −55° C. to 150° C. The thermistor 322 may be a 10kOhm NTC 0603 SMD thermistor available from Murata Electronics, NorthAmerica, Smryma, Ga., for example.

The processing device 330, the heating blocks 282 a, b, c, the resistiveheaters 320, the thermistor 322, and/or the LEDs 288 a, b, c may bemounted to the circuit board 300 by a standard pick-and-place automationdevice conducting standard surface mount technology, in a manner knownin the art. The same pick-and-place device may be used to mount all orseveral of these components, facilitating manufacture of and decreasingthe cost of the board 300 and the analytic device 200.

FIG. 17 is a rear perspective view of FIG. 16. The heating block 282 a,as well as the heating blocks 282 b and 282 c, define a recess 324configured to receive and encompass the resistive heater 320 and thethermistor 322 when the respective heating block is mounted to theprinted circuit board 300. The heating blocks 282 a, 282 b, 282 c may besurface mounted to the circuit board 300 and to the resistive heaters320 and thermistors 322 by thermally conductive epoxy, for example, toprovide intimate thermal contact. This results in accurate temperaturereadings and small thermal inertia.

By providing separate heating blocks 282 a, b, c for each assay tube 234a, b, c in this embodiment, the mass of each heating block 382 a, b, cis decreased, yielding blocks with a high surface area to volume ratio.This decreases the amount of time needed to heat the heating blocks 382a, b, c by the resistive heaters 320 and to cool the heating blocks bythe fan 236 to desired temperatures to perform the assay procedure. Morecomplex heating and cooling schemes are therefore not required. For bestheating, the level of the assay in each assay tube 234 a, b, c is nohigher than the top of the respective heating block 382 a, b, c when theassay tube is in a respective recess 280 a, b, c.

The heating blocks 382 a, 382 b, 382 c may be aluminum as aluminumalloy, such as 6061 which is readily available. The heating blocks 382a, b, c may be formed by machining, for example.

The heating blocks 282 a, b, c have a volume at least as large as thevolume of the fluid to be heated in the assay tubes. In one example, thevolume of fluid in each assay tube 234 a, b, c is from about 5 to about100 microliters, for example. The dimensions of each heating block 282a, 282 b, 282 c may be about 0.25 inches×0.18 inches×0.25 inches, forexample.

FIG. 19 is a rear view of the core 271 showing the printed circuit board300, the assay tubes 234 a, b, c, and the fan 236. A side wall 328, inconjunction with an opposite side wall not shown in this view, guidesthe air from the fan toward the heating blocks 384 a, b, c. The rearside of the board 300 includes a processing device, such as amicroprocessor or a microcontroller for example. In this example, theprocessing device 330 is a microcontroller. The microcontroller iselectrically coupled to and controls the operation of the resistiveheaters 320, the fan 236, and the LEDs 288 a, b, c. The microcontroller330 is also electrically coupled to the thermistors 322 to monitor thetemperatures of the respective heating blocks. The microcontroller 330also communicates with the smartphone 206 to receive instructionsconcerning the assay procedure and to provide information concerning theprocedure to the smartphone wirelessly (via Bluetooth in this example).The microcontroller 330 may also communicate via direct electricalconnection, as discussed above.

Operation of the processing device 330 is controlled by software storedin memory in the analytic unit, which may be part of the processingdevice and/or be mounted to the circuit board 300. The processing device330 of the smartphone 206 provides inputs to the processing device 330,such as temperatures, and instructions, such as turning on and off theresistive heaters, as described below. The microcontroller may be anATmega 32U4 central processing unit, from Atmel, Corporation, San Jose,Calif., on an Arduino electronics platform, for example. A separatesurface mountable wireless communication chip 332 enabling wirelesscommunication between the processing device 330 and the mobileelectronic device 206, may also be surface mounted to the circuit board300. The wireless communication chip may be a Bluetooth® chip, such as aBluetooth® Low Energy System-on-Chip, TI CC2540, from Texas Instruments,Dallas, Tex.

In another example, the resistive heaters 320 are mounted to theopposite side of the circuit board 300 than the heating blocks 282 a, b,c. In this case, the resistive heaters 320 may be thermally coupled tothe heating blocks 282 a, b, c by standard plated through hole vias inthe circuit board 300, as is known in the art.

The battery 238, which powers the analytic unit 202, may be arechargeable battery, for example. A lithium ion or lithium polymerbattery may be used, for example. The analytic unit 202 may include aport to receive a charger plug to recharge the battery by wall power,for example. The PCR device 202 may also be powered by an externalsource of power, such as standard wall power, via a walltransformer/adaptor or other such UL listed device to provide low DCvoltage.

An example of a PCR procedure performed by the analytic device 200 willbe described with respect to a test for Neisseria gonorrhoeae. Thetemperatures and time periods in this example may vary for differenttarget nucleic acid sequences, and for particular analytic devices. Aurine sample is obtained and processed, if required, prior to DNAisolation, in a manner known in the art. The DNA may be isolated inaccordance with the Boom method or other method known in the art by theuser conducting the test or another party, for example. The user of theanalytic device 200 inserts the isolated DNA solution into an assay tube234 a containing lyophilized reagents specific to Neisseria gonorrhoeaefrom a PCR test kit. The assay tube 234 a is shaken and the lyophilizedreagents dissolve in the solution. The user opens the lid 228 andinserts the assay tube 234 a into a recess 280 a in a heating block 282a, for example. A pure water sample is introduced into another assaytube, such as tube 234 b, for example, to serve as a non-templatecontrol, and inserted into the heating block 282 b, for example. Apositive control solution is inserted into another assay tube, such asthe tube 234 c, and inserted into the heating block 282 c, for example.As noted above, the pure water sample should show no fluorescence,unless there is contamination, and the positive control solution shouldfluoresce if the analytic unit 202 performs the PCR procedure correctly,under the control of the smartphone 206. For best heating, the level ofthe assay in each assay tube 234 a, b, c is no higher than the top ofthe respective heating block 382 a, b, c when the assay tube is in arespective recess 280 a, b, c.

The user may then insert (or has previously inserted) a camera enabledmobile electronic device 206, such as a smartphone, into the dockingstation 204. The user opens a PCR App on the smartphone 206 and selectsthe appropriate gonorrhea test protocol. A start button is displayed onthe display 18 and the user may touch the button to start the test.

Wireless or direct electrical connection between the smartphone 206 andthe processing device 330 of the analytic device 202 is confirmed by thesmartphone 206 and the process temperatures are provided by thesmartphone 206 to the processing device, which stores the temperaturesin memory. For this PCR procedure, four temperatures T1, T2, T3, T4 areprovided, where T1=100.0 degrees C., T2=95.0 degrees C., T3=57.5 degreesC., and T4 equals 60.0 degrees C.

Under the control of the smartphone 206, the analytic unit 202 causesthe heating blocks 234 a, b, c to heat to T1 (100.0 degrees C.) byturning on the resistive heaters 320. This starts the initial denaturingphase of the PCR procedure, where DNA strands in this example areseparated to form single strands. When all the heating blocks 234 a, b,c reach the temperature T1, as determined by each thermistor 322, thetemperature is held for a first predetermined Time Period 1 of 6.5seconds.

At the end of the first predetermined Time Period 1, the resistiveheaters 320 are turned off under the control of the smartphone 206,allowing the heating blocks 234 a, b, c to cool to the secondtemperature T2 of 95 degrees C. The temperature T2 is held for twominutes, continuing the denaturing process.

At the end of second predetermined time period T2 of two minutes, theanalytic unit 202 cools the heating blocks 234 a, b, c to thetemperature T3 of 57.5 degrees C., by turning on the fan 236. When thetemperature T3 is reached, the fan 236 is turned off and the heatingblocks 234 a, b, c are held for a second predetermined Time Period 3 ofthree seconds, under the control of the smartphone 206, to start theannealing phase of the PCR procedure.

After three seconds, the resistive heaters 320 are turned on to heat theheating blocks 232 a, b, c to the fourth temperature T4 of 60.0 degreesC. When the fourth temperature T4 is reached, the resistive heaters 320are turned off and the heating blocks 234 a, b, c are held at T4 for afourth predetermined Time Period 4, to start the annealing phase of thePCR procedure. During annealing, the primers attach to a complementarysequence and polymerase joins the dNPT to the 3′ prime end of theprimer, forming a complementary sequence. Annealing continues during thefourth predetermined Time Period 4 of 20 seconds.

Image capture by the rear facing camera 52 of the smartphone 206 takesplace during the fourth predetermined Time Period 4, after the start ofthe Time Period 4. In this example, image capture begins 15 secondsafter the beginning of the fourth Time Period 4 and proceeds for fiveseconds, until the end of the fourth Time Period 4. Image capture may bein a video mode or in a single image mode.

Luminosity data, for example, is derived from the captured images byapplying image processing techniques known in the art by the processingdevice of the smartphone 206, under the control of the PCR App, forexample. In one example, the luminosity values of the images captured ineach cycle may be averaged. The luminosity data may be expressed in theform of a graph that shows the luminosity values from each cycle, overthe entire PCR procedure, as is known in the art. The presence of atarget nucleic acid sequence and the initial concentration of the targetnucleic acid sequence may be determined from the graph, by the PCR Appand/or by the user. The graph may be displayed on the display screen 18of the smartphone, and/or sent to a third party via a network, foranalysis and storage, for example. In another example, the PCR Appprovides an output that the target nucleic acid sequence is present ornot.

The process then returns to the first denaturing step at the firsttemperature T1 and the process is repeated over multiple cycles, such asfor 40 cycles or for a user defined number of cycles, for example. Inthis example, the only difference between the first cycle and thesubsequent cycles is that the second Time Period 2 at 95 degrees C. isonly held for one second.

FIGS. 20-26 show a flowchart 500 of an example of the operation of thesmartphone 206 and the analytic unit 202 in accordance with anembodiment of the invention, based on the gonorrhea PCR proceduredescribed above. The actions of the smartphone 206 or other mobileelectronic device is described in the left column, and the actions ofthe PCR analytic unit 202 are shown in the right column. The flowchartis applicable to the PCR analytic unit 10 of the first embodiment, aswell.

A start command is received by the smartphone 206, in Step 502 in FIG.20. The start command may be entered by PCR App stored on the smartphone206, when or after the PCR App is opened, for example. In response tothe start command, in this example the smartphone 504 sends a Bluetoothsynchronization message to the PCR analytic device 202, in Step 504, toconfirm that there is a Bluetooth connection between the smartphone 206and the analytic unit 202. If the smartphone 206 and the PCR analyticunit 202 are connected via an electrical connection port, the connectionand proper communication can be confirmed by other methods known in theart. The Bluetooth synchronization message may comprise one or moremessages, such as a heartbeat type message, that needs to be confirmedwithin predetermined periods of time, for example. For example, theanalytic unit 202 may need to respond to the synchronization messagewithin 10 seconds. If the Bluetooth synchronization message is receivedin Step 506, the unit 202 sends a message confirming receipt, in Step508. It is noted that this confirmation procedure may be performedcontinuously throughout the PCR procedure. If at any time a confirmationmessage is not received, the assay procedure is aborted.

If the smartphone 206 receives the confirmation message with thepredetermined time, in Step 510, the processing device of thesmartphone, under the control of the PCR App, for example, sends aninitialization command, in Step 512. In this example, the initializationcommand includes four target temperatures T1, T2, T3, T4 to be used inthe PCR process to be performed by the unit 202. The temperatures T1-T4,as well as the predetermined Time Periods, in the method 500 are thesame as the temperatures and time periods discussed above in the PCRprocedure to determine whether Neisseria gonorrhoeae is present. Inother PCR assay procedures, more or fewer temperatures, and differenttemperatures, may be used.

The analytic unit 202 receives the initialization command in Step 514and the processing device 330 stores the temperatures T1-T4 in memory,in Step 516. When temperatures T1-T4 are stored, the processing device330 sends a message to the smartphone 206 that storage of thetemperatures is complete, in Step 518. The smartphone 202 receives thestore temperature complete message in Step 520.

When the store temperature complete message is received by thesmartphone 206, the method 500 continues in FIG. 21 at Step 522, wherethe processing device of the smartphone 206, under the control of thePCR App, sends a heating command to the analytic unit 202. The heatingcommand instructs the unit 202 to heat the heating blocks 282 a, b, c,in this example to the temperature T1.

The processing device 330 of the unit 202 receives the heating commandin Step 524 and retrieves the temperature T1 from memory in Step 526.The processing device 330 then applies a voltage to the resistiveheaters 320 so that they heat the heating blocks 282 a, b, c, in Step528.

The processing device 330 then monitors the temperatures of the heatingblocks 282 a, b, c via the thermistors 322, in Step 530. The processingdevice 330 checks whether T1 is reached, within tolerances, in Step 532.The processing device 330 may check by comparing the current temperaturewith the retrieved temperature T1. In this example, tolerances for thetemperatures T1-T4 may be from about 0.25 degrees centigrade to about0.50 centigrade may be acceptable, for example. If T1 is not reached,then the processing device 330 continues to monitor the temperature inStep 530 and check the temperature in Step 532. The processing device330 waits until all of the heating blocks 282 a, b, c reach thetemperature T1. If T1, and the other temperatures discussed, in thisexample, for each heating block, are not reached within a predeterminedperiod or periods of time, then an error message may be provided to thesmartphone 206. The smartphone 206 can indicate the error to the uservia the display 18 and/or the speaker 22, for example. Error detectionis not indicated in the flowchart 500, but could be readily implementedby one of ordinary skill in the art. When T1 is reached, the processingdevice 330 sends a T1 reached message to the smartphone 206, in Step534.

The smartphone 206 receives the T1 reached message in Step 536. Theprocessing device of the smartphone 206 then retrieves a firstpredetermined Time 1 from memory, in Step 538. The processing devicewaits for the predetermined Time Period 1 to elapse, in Step 540. Theprocessing device checks whether the predetermined Time Period 1 haselapsed, in Step 542. The processing device may check whether the TimePeriod 1 has elapsed by counting down from the predetermined Time Period1 to zero, based on an internal clock, and checking whether zero isreached, in Step 542, for example. If not, the processing device returnsto Step 540 and checks again in Step 542. If it is determined in Step540 that the Time Period 1 has elapsed, the processing device proceedsto Step 544, in FIG. 22.

In Step 544, the processing device sends a command to the unit 202 toallow the heating blocks 282 a, b, c to cool to the temperature T2. Theallow to cool message is received by the unit 202, in Step 546. Inresponse, the processing device 330 of the unit 202 retrieves thetemperature T2 from memory, in Step 548, and turns off the voltage tothe resistive heaters 320, in Step 550. The processing device 330monitors the temperatures of the heating blocks 282 a, b, c, in Step552, and determines whether the temperature T2 is reached, in Step 554.This may be determined as discussed above with respect to Steps 530,532. If the temperature T2 is not reached, the processing devicecontinues to monitor the temperature of the heating blocks 282 a, b, c,in Step 552, and determine whether the temperature is reached, in Step554.

If the temperature T2 is reached, the processing device 330 sends a T2reached message to the smartphone 206, in Step 556. When the smartphone206 receives the T2 reached message, in Step 558, the processing deviceof the smartphone retrieves the second predetermined Time Period 2. Theprocessing device waits for the Time 2 to elapse in Steps 562, 564, asdiscussed above with respect to Steps 540, 542.

When the processing device of the smartphone 206 determines that theTime 2 has elapsed, the method 500 proceeds to Step 566 in FIG. 23,where the processing device of the smartphone 206 sends to the analyticunit 202 a command to cool the heating blocks to temperature T3. Theunit 202 receives the cooling command in Step 568, and the processingdevice 330 retrieves the temperature T3 from memory in Step 570. Theprocessing device 330 turns on the blower to cool the heating blocks 282a, b, c, in Step 572.

The processing device 330 then monitors the temperatures of the heatingblocks 282 a, b, c, in Steps 574 and 576 in the same manner as discussedabove with respect to Steps 552 and 554. When the temperature T3 isreached, the processing device sends a T3 reached message to thesmartphone 206 in Step 578.

The smartphone 206 receives the T3 reached message, in Step 580, and theprocessing device retrieves the third predetermined Time Period 3 frommemory, in Step 582. The processing device then waits for the TimePeriod 3 to elapse, in Steps 584 and 586, as described with respect toSteps 540 and 542.

When the Time Period 3 has elapsed, the method 500 proceeds to Step 588in FIG. 24, where the processing device of the smartphone 206 sends acommand to the unit 202 to ramp the temperature of the heating blocks tothe temperature T4. The ramp command is received by the processingdevice 330 of the unit 202, in Step 590. The processing device 330retrieves T4 from memory, in Step 592, stops the cooling in Step 594 byturning off the blower, in Step 594, and starts heating the heatingblocks 282 a, b, c in Step 596 by turning on the voltage to theresistive heaters 320. The processing device 330 then monitors thetemperature of the heating blocks 282 a, b, c via the thermistors 322,in Steps 598 and 600, as discussed above with respect to Steps 552 and554. When the temperature T4 is reached, the processing unit 330 sends aT4 reached message to the smartphone 206 in Step 602.

The smartphone 206 receives the T4 reached message, in Step 604, and theprocessing device retrieves the fourth predetermined Time Period 4, andthe fifth predetermined Time 5 after the start of the fourth Time PeriodT4, in Step 606. The processing device of the smartphone 206 then waitsfor the Time 4 to elapse, in Step 608 in FIG. 25.

While waiting for the Time Period 4 to elapse, the processing devicechecks whether a fifth predetermined Time T5 is reached in Step 610. Theprocessing device may do this by counting from the start of the fourthpredetermined Time Period T4 to the Time T5.

When the Time Period T5 is reached, imaging starts. The processingdevice sends a message to the unit 202 to turn on the LEDs, in Step 612.The unit 202 receives the message, in Step 614 and the processing device330 provides power to the LEDs 288 a, b, c, in Step 616.

An LEDs on message is then sent to the smartphone 206, in Step 618. Themessage is received by the smartphone 206 in Step 620, and theprocessing device of the smartphone turns on the rear facing camera 52,in Step 622. The camera 52 is on until the end of the fourth Time PeriodT4 is reached. The camera 52 may take a video of the assay, or a seriesof individual images.

When the fourth Time Period T4 is reached in Step 624, the processingdevice turns off the camera 52, in Step 626, and proceeds to Step 630 inFIG. 26.

The smartphone 206 sends a command to the analytic unit 202 to heat theheating blocks to 282 a, b, c to the first temperature T1, to start anew thermal cycle. In this and subsequent cycles, the first temperatureT1 is held for a Time Period 6 different from the first Time Period 1,as discussed above.

The processing device 330 of the analytic unit 202 retrieves thetemperature T1 in Step 630, turns on the voltages to the resistiveheaters 320 in Step 632, and monitors the temperatures of the heatingblocks 382 a, b, c, via the thermistors 322 in Step 634. When thetemperature T1 is reached, in Step 636, the processing device sends a T1reached message to the smartphone 206, in Step 638.

The smartphone 206 receives the T1 reached message in Step 640, and theprocessing device of the smartphone retrieves a sixth predetermined TimePeriod 6, in Step 642, and waits for the Time Period 6 to elapse, inStep 644. When the Time Period 6 elapses, the method 500 proceeds toStep 544 in FIG. 22, to continue the second and subsequent thermalcycles and image capture.

Examples of implementations of embodiments of the invention aredescribed above. Modifications may be made to those examples withoutdeparting from the spirit and scope of the invention, which is definedin the claims, below.

We claim:
 1. A portable analytic device for processing a biologicalsample, comprising: a housing; at least one heating block within saidhousing, wherein said at least one heating block comprises at least onerecess that is configured to receive an assay tube; at least one heatingunit in thermal communication with said at least one heating block,which at least one heating unit provides thermal energy to said assaytube through said at least one heating block; an excitation sourcepositioned to expose said assay tube to an excitation energy; and aprocessing unit comprising a circuit within said housing, wherein saidprocessing unit is operatively coupled to said at least one heating unitand said excitation source, wherein said processing unit is configuredto communicate with a mobile electronic device external to said housing,and wherein said processing unit is configured to: (a) receiveinstructions from said mobile electronic device external to said housingfor processing said biological sample in said assay tube, (b) inresponse to said instructions, (i) direct said at least one heating unitto provide thermal energy to said at least one heating block to provideheat to said assay tube, and (ii) direct said excitation source toexpose said assay tube to excitation energy.
 2. The portable analyticdevice of claim 1, wherein said at least one heating block comprises aplurality of heating blocks and said at least one heating unit comprisesa plurality of heating units, and wherein a given heating block of saidplurality of heating blocks is in thermal communication with a givenheating unit of said plurality of heating units.
 3. The portableanalytic device of claim 1, wherein said at least one heating blockcomprises a first opening disposed on a first side of said at least oneheating block for permitting excitation energy to pass to saidbiological sample within said assay tube, and a second opening on asecond side of said at least one heating block to permit opticaldetection of emission from said biological sample within said assaytube.
 4. The portable analytic device of claim 1, wherein said at leastone heating block comprises a plurality of fins.
 5. The portableanalytic device of claim 1, wherein said instructions comprise atemperature of said at least one heating unit and/or a duration thatsaid at least one heating unit is held at said temperature.
 6. Theportable analytic device of claim 1, wherein said at least one heatingunit comprises a resistive heater, and wherein said at least one heatingunit is thermally cured to said at least one heating block.
 7. Theportable analytic device of claim 1, wherein said at least one heatingunit is at least partially disposed within an additional recess in saidat least one heating block.
 8. The portable analytic device of claim 1,further comprising a communication unit that is configured to providewireless communication between said processing unit and said mobileelectronic device.
 9. The portable analytic device of claim 1, furthercomprising one or more light pipes configured to convey said excitationenergy from said excitation source to said assay tube.
 10. The portableanalytic device of claim 1, further comprising an excitation filter inoptical communication with said excitation source and said assay tube,which excitation filter is configured to limit passage of saidexcitation energy from said excitation source to said biological samplewithin said assay tube to a range of wavelengths.
 11. The portableanalytic device of claim 1, further comprising an emission filter inoptical communication with said assay tube and an optical detector,which emission filter is configured to limit passage of an emission fromsaid biological sample within said assay tube to a range of wavelengths.12. The portable analytic device of claim 1, further comprising a fandisposed at least partially within said housing and operatively coupledto said processing unit, wherein said processing unit is configured toimplement temperature cycling of said biological sample within saidassay tube via said at least one heating block and said fan based, atleast in part, on said instructions from said mobile electronic device.13. The portable analytic device of claim 1, further comprising a dataexchange unit that communicates with said mobile electronic device,wherein said data exchange unit (i) receives said instructions from saidmobile electronic device, or (ii) provides results to said mobileelectronic device upon processing said biological sample.
 14. A methodfor processing a biological sample, comprising: (a) activating aportable analytic device comprising: i. a housing; ii. at least oneheating block within said housing, wherein said at least one heatingblock comprises at least one recess that is configured to receive anassay tube; iii. at least one heating unit in thermal communication withsaid at least one heating block, which at least one heating unitprovides thermal energy to said assay tube through said at least oneheating block; iv. an excitation source positioned to expose said assaytube to an excitation energy; and v. a processing unit comprising acircuit within said housing, wherein said processing unit is configuredto communicate with a mobile electronic device external to said housing;(b) receiving by said processing unit instructions from said mobileelectronic device external to said housing for processing saidbiological sample in said assay tube; (c) in response to saidinstructions, directing said at least one heating unit to providethermal energy to said at least one heating block to provide heat tosaid biological sample within said assay tube; and (d) directing saidexcitation source to expose said biological sample within said assaytube to excitation energy.
 15. The method of claim 14, furthercomprising, subsequent to (d), detecting emission from said biologicalsample within said assay tube, which emission is indicative of apresence or absence, or relative amount, of a target molecule withinsaid biological sample.
 16. The method of claim 14, further comprisingreceiving instructions at said processing unit from said mobileelectronic device, said instructions comprising at least one temperatureat which said at least one heating block is maintained.
 17. The methodof claim 14, further comprising extracting from said biological sampleone or more nucleic acids.
 18. The method of claim 14, wherein saidbiological sample comprises one or more members selected from the groupconsisting of a blood sample, a plant sample, a water sample, a soilsample, and a tissue sample.
 19. The method of claim 14, wherein saidbiological sample contains or is suspected of containing a targetnucleic acid molecule, and wherein said instructions comprise a targettemperature(s) and number of heating and cooling cycles for conductingnucleic acid amplification on said target nucleic acid molecule, underconditions sufficient to yield amplification product(s) indicative of apresence or relative amount of said target nucleic acid molecule. 20.The method of claim 14, further comprising a data exchange unit thatcommunicates with said mobile electronic device, wherein said dataexchange unit (i) receives said instructions from said mobile electronicdevice, or (ii) provides results to said mobile electronic device uponprocessing said biological sample.